Virus production

ABSTRACT

A process for the propagation of hepatitis A virus, which process comprises culturing cells infected with the virus in an aqueous culture medium in which the concentration of sodium chloride is from 30 mM to 170 mM above the isotonic concentration of sodium chloride; and production of a vaccine therefrom.

This is a Rule 60 continuation of of application Ser. No. 08/142,400,filed Dec. 1, 1993.

The invention relates to an improved process for increasing the yield ofHepatitis A virus grown in a culture medium.

BACKGROUND OF THE INVENTION

Hepatitis A virus (HAV) is a member of the Picornaviridae (Melnick, J.L. (1982), Intervirology, 18, 105-106). It is endemic throughout theworld and can cause large outbreaks of disease (W.H.O. 1988, WeeklyEpidemiological Record, 65, 91-92). Protection is afforded byadministration of immune human IgG as at present there is no vaccineavailable. Killed virus vaccines are now under clinical trials but theproduction of a cost-effective vaccine is hampered by the low yields ofvirus obtained from tissue culture systems. Attempts have been made toimprove the virus yield and it has been reported that the cellular RNApolymerase-2 inhibitor 5,6-dichloro1-β-d-ribofuranosylbenzimidazoleincreases yields by 200-300% when incorporated into the culture medium.

A demonstration that HAY would grow in tissue culture (Provost, P. J. &Hillerman, M. R. (1979), Proceeding Society Experimental Biology andMedicine, 160, 213-221) was a considerable advance for the study of thevirus. Since that time strains of hepatitis A have been developed thatgrow more rapidly and to a higher titre than the original isolates.However, compared to other picornaviruses, the yields are still low andthe time to obtain maximum yield is long. There are reports that the useof host enzyme inhibitors can increase the yield of HAV (Widdell, A.,Hansson, B. G., Nordenfelt, E. and Oberg, B. (1988), Journal of MedicalVirology, 24, 369-376), however these are expensive and are unlikely tobe cost effective for virus production for the preparation of vaccines.

SUMMARY

According to the present invention, there is provided a process for thepropagation of hepatitis A virus, which process comprises culturingcells infected with the virus in an aqueous culture medium in which theconcentration of sodium chloride is from 30 mM to 170 mM above theisotonic concentration of sodium chloride. Thus, an improved process isprovided for increasing the yield of HAV which is simple andinexpensive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the rate of production of HAV in media containing andmedia not containing excess sodium chloride.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The concentration of sodium chloride in the medium may be from 50mM to150mM greater than the culture's isotonic concentration of sodiumchloride. A preferred concentration is from 80mM to 120mM, mostpreferably 100mM, above the isotonic concentration of sodium chloridefor the culture. Typically, the isotonic concentration of sodiumchloride is approximately 150mM for media in which cells infected withhepatitis A virus are grown.

If the isotonic concentration of sodium chloride for a culture is known,the absolute concentration of sodium chloride in the culture medium maybe deduced. Hepatitis A virus may therefore be grown by the presentprocess in an aqueous culture medium in which the absolute concentrationof sodium chloride is from 180mM to 320mM. Suitable absoluteconcentrations of sodium chloride in a culture medium are from 200mM to300mM, preferably from 230mM to 270mM. A particularly useful absoluteconcentration of sodium chloride in a culture medium is about 250mM.

The concentration of sodium chloride in the culture medium may be raisedabove the isotonic concentration by adding aqueous sodium chloride theculture medium. The additional sodium chloride may be added to theculture before, simultaneously with or after infection of cells inculture with hepatitis A virus. Any appropriate strain of hepatitis Avirus may be employed. Advantageously, an aqueous solution of sodiumchloride is added to the culture medium from 12 to 48 hours, suitablyabout 24 hours, after infection of the cells in the culture with thevirus.

Cells which are suitable for use in the present process are typicallycells of cell lines which will support the growth of hepatitis A virusand, in particular, of cell lines which are acceptable for theproduction of vaccines. However, any type of cell culture system inwhich the virus is capable of replicating may be employed. The cellculture system may therefore be formed of primary, continouslycultivated or transformed cells. The cell system may be derived fromkidney or liver of human or nonhuman primate origin.

Examples of suitable cell culture systems include those derived fromfetal or new born kidney cells from rhesus monkey, cynomolgus monkey,marmoset or cercopithecus monkey, or diploid fibroblast cells derivedfrom human or non-human primate lung tissue such as, for example, WI-38or MRC-5 cell lines (US-A-4301249); Vero cells (US-A-4783407); humanliver tumor lines PLC/PRF/5 or Hep. G2 (US-A-4721675); or BS-C-1 cells.

A suitable strain of hepatitis A virus is the HM 175 strain, inparticular its 18F isolate. "The origin of the HM 175 strain ofhepatitis A virus" was described by Gust, Lehmann, Crowe, McCrorie,Locarnini and Lucas (Journal of Infectious Diseases, Vol. 151, No. 2,365-67). The adaptation of this virus to grow in tissue cultures isdescribed in "Primary isolation and serial passage of hepatitis A virusstrains in primate cell cultures" by Binn, Lemon, Marchwicki, Redfield,Gates and Bancroft (Journal of Clinical Microbiology, Vol. 20, No. 1,28-33).

The process may take place in any type of cell culture medium in whichthe cells infected with the virus are capable of growing. The medium mayfurther comprise nutrients required for the growth of the cells,generally a source of assimilable carbon, a source of assimilablenitrogen and, optionally, mineral salts. An example of a particularlysuitable cell culture medium is Eagles Minimum Essential Mediumsupplemented with foetal calf serum.

The cells may be infected with the virus by any conventional method. Theconditions under which the infected cell lines are propagated dependupon the nature of the cell line used. Advantageously, the infectedcells are incubated at the temperature which is known to produce maximumgrowth rate. A cell culture may be incubated at 25° C. to 40° C., forexample from 34° C. to 37° C. Cell growth may be sustained until thecytopathic effect is complete. Culture of the cells infected with thevirus in the aqueous culture medium containing additional sodiumchloride may be grown for 2 to 4 days.

The virus obtained by the process of the present invention may beisolated, purified, and inactivated. Inactivated Hepatitis A thusprepared may be formulated with a pharmaceutically acceptable carrier ordiluent in order to produce a vaccine therefrom. Accordingly, theinvention also provides a vaccine comprising a pharmaceuticallyacceptable carrier or diluent, and as an active ingredient, inactivatedhepatitis A virus produced by the present process.

The following Example illustrates the invention. In the accompanyingdrawing, the Figure illustrates the rate of production of HAV in mediacontaining (x--x) and media not containing ( -- ) excess sodiumchloride. The x-axis denotes days post infection. The y-axis denotesradioimmunoassay counts per minute.

EXAMPLE Cell Culture

BS-C-1 cells were obtained from the American Type Culture Collection(ATCC CCL 26) and used between passages 53 and 75 (Hopps et al. (1963),Journal of Immunology, 91 416-424 and Lemon et al. (1983), Journal ofClinical Microbiology, 17 834-839). The cells were grown in 1969 medium(Seefried, A., Healy, G. M., and Macmorine, H. G., (1970), JugoslavenkaAkademija Znanosti I Umjetnosti (Symposium on human diploid cells) andHealy, G. M., Teleki, S., Seefried, A., Walton, M. J., and Macmorine, H.G., Applied Microbiology, (1971), Vol. 21, No. 1, 1-5) containing 10%foetal calf serum (FCS).

Virus Growth

Confluent BS-C-1 cell monolayers were infected with virus harvest togive approximately 1 to 10 radioimmunofocus units/cell (Lemon et al.,(1983) Journal of Clinical Microbiology, 17, 834-839) of HAV per cellfor 1 hr at room temperature. The virus used was the 18F tissue cultureadapted isolate of the HM175 variant of HAV provided by Dr. S. Lemon.The sheets were washed in Eagles Minimum Essential Medium (MEM) andincubated at 34° C. in Eagles MEM containing 2% FCS. Differentconcentrations of sodium chloride (NaCl) were added, usually 24hr later.At various time intervals after infection the flasks were placed at -20°C.

Preparation Of Virus Antigens

The cells were subjected to three cycles of freeze-thawing. Cell debriswas pelleted by low speed centrifugation and resuspended in 10 mM TRIS,10mM NaCl, 1.5 mM MgCl₂, 1% Nonidet P40 and 0.5% alkyl dimethylaminebetaine (Empigen, Albright and Wilson Chemicals). The suspension wasincubated for 1 hr at room temperature. The resulting suspension wasclarified by low speed centrifugation and the supernatant was added tothe supernatant obtained from the freeze-thawed cells. This was used forradioimmunoassay (RIA) or radioimmunofocus assay (RIFA) directly.

Alternatively, the virus solution was made with 100 mM TRIS pH 7.6, 100mM NaCl, 3mM EDTA and sarkosyl added to 1%. After 1 hour at 37° C. thevirus was pelleted by centrifugation at 100,000 x g at 5° C. for 18 hrs.The virus pellet was resuspended in 200ml 100mM TRIS pH 7.6 and 100mMNaCl and used for RIA or RIFA.

Radioimmunoassay (RIA)

RIA was done as described by Lemon, et al., (1982), J. Medical Virology,10, 25-36. Briefly, virus was captured using an anti-HAV polyclonalantiserum. After washing, the captured antigen was detected usingpolyclonal anti-HAV ¹²⁵ I IgG.

Radioimmunofocus assay (RIFA)

HAV infectivity was determined by RIFA (Lemon et al., (1983), J.Clinical Microbiology, 17, 834-839). Virus was absorbed for 1h at roomtemperature and the cells then overlayed in Eagles MEM supplemented with2% FCS and containing 0.5% agarose. Cells were incubated under 5% CO₂for 7 days at 35° C. After acetone fixation of the cell sheet, foci ofvirus replication were detected by reaction with ¹²⁵ I anti-HAYpolyclonal IgG followed by autoradiography.

RESULTS

The addition of 100 mM NaCl to growth medium consistently gave a morerapid cytopathic effect (CPE). Usually by 7 days post-infection CPE wascomplete although this was not observed in control cultures even at 10days post-infection. Table 1 shows the yield of HAV, as measured byradioimmunoassay. There was an apparent increase in yield both in theinitial harvest and in the material concentrated by ultracentrifugation.

                  TABLE 1                                                         ______________________________________                                                            Concentrated by                                           Harvest             ultracentrifugation                                              Con-                 Con-                                              Dil*   trol   +100 mM NaCl  trol +100 mM NaCl                                 ______________________________________                                        1/1    153    379           744  1788                                         1/10   204    252           264  359                                          1/100  120    142           179  179                                          ______________________________________                                         *the dilution 1/1 in Table 1 is the combination of virus in cell              supernatant plus that removed from the cells by detergent. The material       used to infect the cell monolayer was the virus in the supernatant only,      and would be about 50% of the total virus. Dilutions 1/10 and 1/100 are       10fold and 100fold dilutions of the 1/1 dilution respectively.           

To determine the optimum concentration of sodium chloride for enhancingthe virus yield, cells were infected with virus and after 24hrs variousconcentrations of sodium chloride were added. Cells were harvested 7days later and the virus yield determined by RIA and by RIFA. Theresults are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        Extra NaCl (mM)                                                                             RIA cpm (-Bg)                                                                              RIFA                                               ______________________________________                                         0             64          5 × 10.sup.5 /ml                              50           113          8 × 10.sup.5 /ml                             100           169          1.2 × 10.sup.6 /ml                           150           134          7 × 10.sup.5 /ml                             200            30          1 × 10.sup.5 /ml                             250            11          3 × 10.sup.3 /ml                             ______________________________________                                    

Growth Curve of HAY with or without Extra Salt

Since one effect of hypertonic NaCl was to increase the rate of the CPEand because the previous experiments had been terminated at 7 dayspost-infection, it was considered important to determine whetherelevated levels of NaCl increased the absolute yield or simply increasedthe rate at which HAY was produced. 25 cm flasks were infected with 10radioimmunofocus units per cell and incubated with or without extrasodium chloride (100 mM), which was added immediately post-infection.Flasks were harvested at 24 h intervals and the virus extracted andconcentrated by ultracentrifugation. Elevated concentrations of NaClappeared to make little difference to the rate of virus production (FIG.1). The greatest increase was between day 2 and day 3 in both cases,maximum yield being obtained between day 3 and 4. Although there was anincrease in yield with additional NaCl, this enhancement wasconsiderably lower than in previous experiments. This may be related tothe fact that in earlier experiments the extra NaCl was added 24 hrpost-infection whereas in this experiment it was added immediately afterinfection.

I claim:
 1. A process for the propagation of hepatitis A virus, whichprocess comprises culturing cells infected with the virus in an aqueousculture medium in which the concentration of sodium chloride is from 80mM to 170 mM above the isotontc concentration of sodium chloride.
 2. Aprocess according to claim 1, wherein the concentration of sodiumchloride is from 80mM to 120mM above the isotonic concentration ofsodium chloride.
 3. A process according to claim 2, wherein theconcentration of sodium chloride is about 100mM above the isotonicconcentration of sodium chloride.
 4. A process according to claim 1,wherein an aqueous solution of sodium chloride is added to the culturemedium from 12 to 48 hours after infection of the cells in the culturewith the virus.
 5. A process according to claim 1, wherein the cellculture comprises BS-C-1 cells.
 6. A process according to claim 1,wherein the culture medium comprises Eagles Minimum Essential Medium andfoetal calf serum.
 7. A process according to claim 1, wherein the saidculturing is effected for from 2 to 4 days.
 8. A process according toclaim 1, which further comprises recovering the hepatitis A virus thusgrown.
 9. A process according to claim S, which further comprisesinactivating the recovered hepatitis A virus.
 10. A process for thepreparation of a vaccine against hepatitis A virus which processcomprises culturing cells infected with the virus in an aqueous culturemedium in which the concentration of sodium chloride is from 30mM to 170mM above the isotonic concentration of sodium chloride, recovering thehepatitis A virus thus grown, inactiveting the recovered hepatitis Avirus and formulating the inactivetad hepatitis a virus with apharaceutically acceptable carrier or diluent.
 11. A process accordingto claim 10, wherein the concentration of sodium chloride is from 80 mMto 120 mM above the isogonic concentration of sodium chloride.
 12. Aprocess according to claim 11, wherein the concentration of sodiumchloride is about φmM above the isogonic concentration of sodiumchloride.
 13. A process according to claim 10, wherein an aqueoussolution of sodium chloride is added to the culture medium from 12 to 48hours after infection of the cells in the culture with the virus.
 14. Aprocess according to claim 10, wherein the cell culture comprises Be-C-1cells.
 15. A process according to claim 10, wherein the culture mediumcomprises Eagles Minimum Essential Medium and foetel calf serum.
 16. Aprocess according to claim 10, wherein the said culturing is effectedfor from 2 to 4 days.